Cancer is a complex heterogeneous disease with molecular, morphological and clinical differences that exist both between and within tumors. While histopathologic features of tumor cell morphology, invasiveness and metastasis remain the “gold standard” for diagnosis and staging of cancers, molecular profiles of neoplastic cells based on DNA, mRNA, and/or protein alterations are rapidly being developed and utilized not only to augment diagnosis, but to provide new therapeutic measures. Of these profiles, the pattern of protein expression based on cell surface markers is the most functional, holding the potential to offer direct correlations between cancer cell “portraits” and therapeutic responses to anti-cancer drugs.
Several strategies have been implemented to date for identification of distinctive protein profiles for a given cancer. For example, separation of proteins on two-dimensional gels has been used widely for protein expression analysis in research laboratories for many years. However, such analyses are unlikely to be used routinely in a clinical setting due to their complexity and the absence of 2-D protein databases of different tumor types. A peptide microarray method was proposed recently by Aina et al, “Therapeutic Cancer Targeting Peptides,” Biopolymers 66:184-199 (2002), for identification of cell surface binding profiles of cancer cells. With this technique, cell-specific peptides isolated by selection from a one bead-one compound (OBOC) combinatorial library were linked to a polystyrene slide in a microarray format and used to detect a binding profile of human T-lymphoma cells. While the method seems promising for profiling cancer cells derived from individual cancer specimens, the diversity of OBOC libraries is relatively low and could be a limiting factor for their broad applications.
Invented less than 20 years ago, phage display technology is now well known in the art, and is used to produce valuable targeting peptides to a variety of cell types, both in vitro and in vivo. Phage display libraries are heterogeneous mixtures of billions of phage clones. Within a library, each clone carries a different foreign DNA insert and, therefore, displays the corresponding unique peptide on its surface. Different types of phage display libraries exist, depending upon the size of the insert, gene location of insert (resulting in pIII or pVIII phage display), structure of the displayed foreign peptide, and number of copies expressed on the surface of the phage. Importantly, the diverse and complex nature of random peptide libraries have the capacity to provide unique peptide sequences for any target receptor molecules, including those that are well-described and those that are previously undetected. This feature is especially important in ligand development for anti-cancer strategies, since tumors are composed of heterogenic cells that express different levels of cell-specific markers in the majority of patients.
Discovered in early 1980's, antimicrobial lytic peptides have been and continue to be studied extensively as a source of new anti-infective and recently anti-cancer agents. Antimicrobial peptides are a large group of gene-encoded molecules produced by almost all eukaryotic organisms including plants, insects, amphibians, and mammals. They are activated shortly after infection as part of the innate immunity of these species and rapidly destroy a broad range of invading microorganisms. In mammals, the major families of antimicrobial lytic peptides are the defensins and the cathelicidins that provide a first line of defense against pathogens. They are found in many tissues exposed to microbes such as mucosal epithelial surfaces and skin, as well as in professional phagocytes.
At present, thousands of native and de novo designed antimicrobial peptides are known. For example, an updated list of antimicrobial peptides from domesticated animals can be found in Brogden et al, “Antimicrobial Peptides in Animals and Their Role in Host Defences,” International Journal of Antimicrobial Agents 22:465-478 (2003). Sequences of over 750 eukaryotic antimicrobial peptides have been reported and can be obtained from protein databases. Many of antimicrobial peptides are relatively short (15-40 amino acids), cationic (carry a positive net charge), alpha-helix-forming, amphipathic (with hydrophobic residues distributed on one side of the helical axis and cationic residues on the other) molecules. Because of these properties, they attack similar targets, which are the bacteria phospholipid membranes with highly negatively charged outer surfaces determined by high content of anionic phospholipids. These peptides perform their antimicrobial function using a common general mechanism. The hydrophilic, cationic part of the peptide is proposed to initiate electrostatic interaction with the negatively charged components of the bacterial membrane. The hydrophobic portion of the peptide then is inserted into and permeates the membrane causing membrane disintegration.
Selective toxicity of microbial peptides is thought to be due to the composition and structure of bacterial membrane, which maintain large transmembrane potentials and has a higher content of anionic phospholipids on its outer leaflet. In contrast, mammalian cell membrane is composed of zwitterionic (neutral) phospholipids and cholesterol that prevent interaction with the peptides and help to avoid host tissue damage. Some bactericidal peptides were found to be cytotoxic for mammalian cancer cells, Papo et al, “New Lytic Peptides Based on the D,L-amphipathic Helix Motif Preferentially Kill Tumor Cells Compared to Normal Cells,” Biochemistry 42:9346-9354 (2003). While the mechanism of selectivity is not quite clear, it is believed that it can be partially explained by the differences in normal and cancer cell membrane composition. Similar to bacteria, cell membranes of tumor cells contain negatively charged phospholipids (3-9%). Interestingly, eukaryotic mitochondrial membrane is very similar to prokaryotic cytoplasmic membrane and can be a target for antimicrobial peptides as well.
In spite of the fact that some native cationic antimicrobial lytic peptides are more toxic to cancer cells than to normal mammalian cells, there are several limitations that prevent their application in cancer therapy. The use of native all L-amino acid antimicrobial peptides in vivo is limited in part because of enzymatic degradation and binding to components in serum. Additionally, they possess lytic activity toward blood cells. As a solution, Papo et al, “A Novel Lytic Peptide Composed of DL-Amino Acids Selectively Kills Cancer Cells in Culture and In Mice,” Journal of Biological Chemistry 278:21018-21023 (2003) proposed de novo designed diastereomeric peptides (composed of both L- and D-amino acids). These peptides were derived from potent lytic peptides by replacing a few L-amino acids with their D-enantiomers. The resulting diastereomeric peptides lost their cytotoxic effect against normal mammalian cells, but preserved both their antibacterial activity and their ability to increase the permeability of negatively charged phospholipid membranes, including cytoplasmic membranes of cancer cells. The authors found that the selective activity of a 15-mer diastereomeric peptide toward cancer cells is a consequence of selective binding, mainly governed by electrostatic interactions between this cationic peptide and anionic phospholipids of cancer membranes. A short time after binding about 10 min), the cells died as a result of acute injury characterized by swelling and bursting, suggesting necrosis. ATR-FTIR studies showed that the peptide initially binds onto the surface and then is inserted into the membrane, destabilizing the membrane structure. At the same time, LD-amino acid peptides are not lytic toward normal mammalian cells.
To produce potent peptides with specific anti-cancer activity, several research groups constructed peptide molecules with two domains. One of the domains is a cell-targeting peptide, which is designed to guide the whole molecule to the target (tumor) cell, allowing cell-specific receptor-mediated internalization. The second domain is a lytic peptide which, after internalization, is able to destroy mitochondrial membrane and cause cell apoptosis. The whole peptide molecules are short, consist of 20-25 amino acids, and can be synthesized chemically with ease. Chen et al, “RGD-Tachyplesin Inhibits Tumor Growth,” Cancer Research 61:2434-2438 (2001), examined a preparation of tachyplesin (17 amino acid antimicrobial peptide present in leukocytes of the horseshoe crab) that was linked to RGD peptide. RGD peptide is known to bind to integrins and thereby can facilitate internalization of tachyplesin. They found that this synthetic peptide with RGD-tachyplesin sequence could inhibit the proliferation of TSU prostate cancer cells and B16 melanoma cells in a dose-dependent manner in vitro and reduce tumor growth in vivo.
Ellerby et at, “Anti-Cancer Activity of Targeted Pro-Apoptotic Peptides,” Nat. Med. 5:1032-1038 (1999), conjugated a short (14 amino acids) cationic lytic peptide with tumor-homing domain, either CNGRC (SEQ ID NO:1) or ACDCRGDCFC (SEQ ID NO:2). It was shown that the resulting peptides with dual function exhibited antitumor activity through their ability to target tumor cells and trigger apoptosis via disruption of mitochondrial membranes. Sequences of the tumor cell-targeting peptides used in this study were identified from screening of phage display libraries. Using phage display, Kelly and Jones, “Isolation of a Colon Tumor Specific Binding Peptide Using Phage Display Selection,” Neoplasia 5:437-444 (2003), identified peptides that recognize colon carcinoma cells. One of the peptides was coupled to the mitochondrial toxin (the same as in Ellerby et al, “Anti-Cancer Activity of Targeted Pro-Apoptotic Peptides,” Nature Medicine 5:1032-1038 (1999)) and added to HT29 colon carcinoma cells. As determined by MTT assay, HT29 cell viability after incubation with the peptide decreased in a concentration dependent manner.